Cbfa1 mediates mesenchymal cell commitment to an osteochondroprogenitor lineage and supports continued differentiation and function in committed osteoblasts. However, definitive downstream mediators of Cbfa1 that critically impact osteoblast differentiation and bone formation have yet to be described. Ne1 (a protein strongly expressed in neural tissue encoding epidermal growth factor like domain) was first identified in chicken embryos. The preliminary data shows that Nell-1 (Ne1-like molecule-1) in vivo is associated with normal and pathological suture fusion, while Nell- 1 in vitro is associated with increased osteoblast differentiation and mineralization. Furthermore, the human Nell-1 promoter contains three osteoblast-specific cis-acting element 2 (OSE2) response elements for Cbfa1 and that Nell-1 overexpression mice can functionally compensate for some aspects of intramembranous bone deficiency in heterozygous Cbfa1 deficient mice. These data have led to the hypothesis that Nell-1 is a central downstream target of Cbfa1 that supports continued differentiation and function in committed osteoblasts and proper Nell-1 expression is required for normal membranous bone growth and formation. To test this hypothesis, this proposal will analyze the following: 1) the regulation of Nell-1 transcription by Cbfa1 during osteoblast formation and function and by Cbfa1 through OSE2 response elements; 2) the functional compensation of Cbfa1 deficiency by Nell-1 in vivo and in vitro; and 3) the effects of targeted Nell-1 disruption in vivo. If Nell-1 is proven to be a critical mediator of intramembranous bone formation, it can further aid in understanding the development of intramembranous vs. endochondral bone. In addition, since sutures are major sites of calvarial intramembranous bone growth the close association of Nell-1 with calvarial sutures can also impact present knowledge on cranial vault development. Lastly, Nell-1 can be tremendously useful as a clinical tool to inhibit or induce intramembranous bone growth, with the secretory nature of Nell-1 making this utility even more feasible. Controlled inhibition of intramembranous bone growth by anti-Nell-1 strategies in CS patients may offer a less invasive, biologically based, therapeutic alternative to radical surgical craniofacial reconstruction. In addition, the ability of Nell- 1 to induce calvarial osteoblast differentiation may be clinically applied to situations where membranous bone formation and regeneration are desirable (e.g., cleft lip or cleft palate repair and craniofacial distraction osteogenesis).